Fig 1.
General methodology employed in the study.
The multi-epitope subunit vaccine design pipeline employed in this study was categorized into four groups, as delineated by boxes: protein retrieval, epitope mapping, vaccine design, and vaccine evaluation. Figure created using BioRender.com.
Table 1.
Predicted LBL epitopes, their positions in protein sources, and scores from prediction and physicochemical tests.
Table 2.
Predicted CTL epitopes, their positions in protein sources, and scores from physicochemical tests.
Table 3.
Predicted HTL epitopes, their positions from protein sources, and scores from physicochemical tests.
Table 4.
HTL epitopes associated with at least one cytokine induction.
Table 5.
Final list of CTL and HTL epitope components of the vaccine and the MHCs in which it demonstrated superior binding affinities.
Fig 2.
Relative binding free energies and energy decomposition analyses for CTL epitopes (A-C) and HTL epitopes (D-F), along with their respective control complexes. Epitope complexes are colored blue, whereas control complexes are represented in gray. Two sets of free energy calculations were performed using MM/GBSA method: pre-molecular dynamics using Hawkdock (A,D) and post-molecular dynamics using the gmxMMGBSA tool (B,E). Energy decomposition within the MHC residues involved in peptide binding (C,F).
Table 6.
Endogenous adjuvants used in the study.
Fig 3.
Comparison of the immune simulation profiles of the four vaccine constructs with different adjuvant formulations.
Each formulation was differentiated using distinct colors. The gray line on the 28th day indicates the second immunization while the gray line on the 56th day indicates the third immunization. Graphs: (A) Antibody titers. (B) Interferon-γ concentration. (C) Cytotoxic T-lymphocyte (TC). (D) Helper T-lymphocyte (TH) populations. (E) B-lymphocyte populations.
Fig 4.
Graphical representation of the final vaccine construct components.
This displays the identities and arrangement of the epitopes, adjuvants, and linkers.
Table 7.
Physicochemical properties of the vaccine constructs with different adjuvant formulations.
Table 8.
Physicochemical properties of the NiV multi-epitope subunit vaccines.
Fig 5.
Comparison of the immune simulation profiles of the three multi-epitope subunit NiV vaccine designs.
The straight blue line represents the NiV vaccine design of this study, while broken lines represent NiV vaccine designs from other studies, each differentiated by a distinct color. The gray line on the 28th day indicates the second immunization while the gray line on the 56th day indicates the third immunization. Graphs: (A) Antibody titers. (B) Interferon-γ concentration. (C) Cytotoxic T-lymphocyte (TC). (D) Helper T-lymphocyte (TH) populations. (E) B-lymphocyte populations.
Fig 6.
Comparison of the quality of the tertiary structure model predicted by (A) Alphafold, (B) D-ITASSER, and (C) Robetta. The adjuvant, epitope groups, and linkers are differentiated by distinct colors. In ProSA-web, a structure within the z-scores range indicates an accurate and reliable structure. A high-resolution structure in ERRAT produces 95% quality or higher and 91% for lower resolutions (2.5–3.0Å). A good quality structure in Procheck generally contains 90% residues in the most favorable regions.
Fig 7.
Conformational B-lymphocyte epitope regions within the vaccine construct predicted by the Ellipro tool.
The darker the area, the higher the epitope score.
Fig 8.
Molecular docking and dynamics analyses of the vaccine construct and TLR4-MD2.
Two conformations of the binding, Complex 3 (top) and 7 (bottom) were shown. Figures: (A) Model of the complexes. (B) Model showing residues involved in the interaction of the complexes. (C) Per-residue binding free energies of TLR4 and MD2. (D) RMSD graph of the complexes following 1000 ns molecular dynamics simulations.
Fig 9.
Cysteine-mutated residues within the vaccine construct.
Residues suitable for mutation were identified using Disulfide by Design 2.0.
Table 9.
Residues within the vaccine construct mutated to cysteine.
Fig 10.
Cloned vaccine construct in a pET28(a)+ vector.
The codon sequence is indicated in red while the plasmid backbone is shown in black.